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Title: Oral mucosa-nanoparticle interactions and uptake pathways in formulation excipient profiling
Author: Best, Mark
ISNI:       0000 0004 5348 4731
Awarding Body: University of Brighton
Current Institution: University of Brighton
Date of Award: 2014
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Nanomaterials are generally defined as chemical substances or materials that contain particles with one or more dimensions less tl1an 100 nanometres in size. They may be either engineered or naturally occurring, but have unique properties due to a vastly increased surface area to volume ratio when compared to non-nano (bulk) materials. This provides the potential for the development of a wide range of enhanced formulations with superior efficacy including applications in oral health care .. As the properties of a material change at the nano-scale, there are concerns that the toxicological profile of these materials may also change. Size is only one factor; changes in shape, surface chemistry, chemical composition, porosity and solubility all contribute to the overall biological toxicity profile of a nano-scale ingredient. Established links between the specific properties of a nanomaterial and toxicity are not well understood, leaving an important data gap in the literature. The purpose of this work was to utilise in vitro oral epithelial models for the assessment of safety profiles of nanomaterials for applications in next generation oral care products. Four commercially sourced nanomaterials were analysed, alongside respective bulk counterparts already found within oral care product formulations. These nanomaterials comprised of two nano-zinc oxides (ZnO), silicon dioxide (SiOz), titanium dioxide (TiOz) and hydroxyapatite (Cas(OH)(P04)'). Comprehensive characterisation of each material was carried out using a range of analytical techniques to identify any structure-function relationships in vitro. Initial toxicity screening experiments were conducted using a non-keratinised oral epithelial cell monolayer (H376 cell line) with both cell viability and lysis analysed using MTT and LDH assays respectively. Materials were investigated further using two 3-dimensional tissue models representative of the main tissue types constituting the human oral mucosa: non-keratinised buccal (RHO) and keratinised gingival (GIN-lOO) models. Nanomaterial uptake in the models was investigated using confocal microscopy with a styrl dye (FM 1-43). This led to the development of a novel, high throughput fluorescent assay as a potential method for screening nanoparticle-uptake. Results highlighted the complexities involved with nano-characterisation in biological media using current techniques. A wide variety of particle shapes and sizes were recorded between different nanomaterials, with results being dependent upon the sample preparation steps and specific methods of analyes used. These disparities represent the current challenges experienced by both researchers and regulators of nanotechnology at the present time. ZnO \vas observed to be the most cytotoxic material during monolayer screening, at concentrations exceeding 0.3125% w/v when delivered in protein-free media. Differences between bulk and nanomaterial properties were recorded for all the materials, except for Ti02, but these did not necessarily transfer to effects seen in the more representative 3-D models. Cytotoxicity results from both R1--10 and GIN-lOO models exemplified the disparity between sensitivity of monolayer and the natural stratified tissue structure of human oral mucosa. Keratinised gingival tissue models showed slgnificantly greater durability over the less robust buccal model, in both cytotoxicity assays and IL- lcx cytokine response. Of all materials examined, cellular uptake was only observed for nano-Si02. This was the only material detected trafficking inside the cell using the FM 1-43 styryl dye assay, with confocal data serving to verify the analysis of nanoparticle Internalisation using fluorescence. In conclusion, nanomaterials pose considerable difficulties during formulation and analysis in health care products. The risk of potential uptake and bioaccumulation or translocation to particularly sensitive areas of the body also requires further investigation. Nanomaterials have to be assessed on a case by case basis, and robust/consistent regulatory strategies developed to enable industry to produce and market novel but safe nanoparticle containing formulations. Risks to human health may be less of a hazard when applied to fully functioning healthy human tissue, especially in comparison to existing bulk material effects and current, accepted irritant ingredients (e.g. Sodium lauryl sulphate).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: B000 Health Professions